1. Field of the Invention
The present invention relates to methods of evaluating plant protection and, more particularly, to a method of evaluating the efficacy of a plant protection mechanism or other plant characteristic for resistance against an insect.
2. Description of Related Art
Many insect pests of crop plants cause damage which can be difficult to measure precisely. Because this damage can be difficult to measure, it can also be difficult to determine whether a particular plant protection mechanism is effective in reducing damage from insect pests.
For example, the western and northern corn rootworm, Diabrotica virgifera virgifera and D. barberi, respectively, are perennial insect pests of corn across most of the Corn Belt. Both species are univoltine and overwinter as eggs in the soil, typically of cornfields. The larvae of such pests are often the most damaging insect stage. Larval feeding on the roots reduces crop yield by limiting transport of water and nutrients from the soil and increasing plant susceptibility to lodging.
Historically, plant protection mechanisms used against these pests have included crop rotation and/or synthetic insecticides applied to the soil at planting time. The protection provided by insecticides against corn rootworm larvae has traditionally been evaluated subjectively in field plots by visually scoring the amount of root damage relative to an unprotected control. Root damage ratings have generally been adopted as the standard evaluation tool for the efficacy of plant protection mechanisms against corn rootworm because root ratings provide direct evidence of the measure of protection. However, it has been inefficient or impractical to relate the direct effects of a plant protection mechanism on the insects to the root injury expected in a field setting. Although root ratings do provide a useful tool for categorizing the efficacy of protection mechanisms against corn rootworm, such methods may lack statistical validity, and study results may be difficult to interpret because of, for example, variability associated with interactions between the insect, the protection mechanism, maize genetics, and the environment.
One example of a plant protection mechanism is a transgene (such as, for example, a polynucleotide encoding an insecticidal protein) that is incorporated into a plant so as to, for example, protect a maize plant from corn rootworm. Such a plant protection mechanism may have unique biological, physiological, and/or regulatory characteristics that may make evaluation of its efficacy relatively more complex with respect to, for example, other plant protection mechanisms. By “transgene” is intended a gene or polynucleotide that has been introduced into the plant genome by human intervention, such as by transformation and/or by breeding.
One of the first issues in the development process for such plant protection mechanisms is identifying insecticidal proteins with suitable bioactivity properties against the target pest. Historically, evaluation of such proteins was accomplished using an artificial diet bioassay for the insects wherein the diet was treated with purified insecticidal protein and the evaluation was based on the number of insects that died during the bioassay. However, using the death of the insect as a measure of efficacy has several drawbacks. First, death is a relatively crude measurement of the impact of a particular treatment. Also, death may be due to factors other than the insecticidal protein. With soil-inhabiting insects like corn rootworm, diet contamination is an important factor that can limit the interpretation and precision of bioassay results. Additionally, the cost of such assays is typically high due to the need for purified insecticidal protein. Further, protein bioactivity observed in a diet bioassay may not necessarily correlate to efficacy of a protection mechanism in plants expressing the insecticidal protein under field conditions.
Another important step in the development process of a plant protection mechanism which is a transgene is that the efficacy of several independent transformation events are typically evaluated. A transformation event results from the process by which a transgene is inserted into the plant genome, and transformation events typically vary in their expression levels and consequently in the efficacy of a plant protection mechanism provided thereby. Accordingly, it is standard in the art to evaluate the efficacy of at least several independent transformation events by evaluating the trait in plants that contain a transformation event.
In such evaluations of efficacy for traits intended to provide protection against corn rootworm, root damage ratings typically are used to identify events that may have levels of efficacy suitable for commercial use. However, measurements of root damage have several drawbacks, including a requirement for large sample sizes in order to increase precision. The large sample sizes required are often difficult to obtain from a limited number of transgenic plants. There is also inherent variability associated with the root rating measurements, and these factors both act to limit predictability when attempting to discriminate subtle differences in efficacy between transformation events. Thus, for example, it would be helpful to have a relatively sensitive bioassay in order to distinguish and characterize differences among: transgenic plants comprising different insecticidal proteins; differences among transformation events using the same trait; differences between the same event expressed across a number of genetic backgrounds; and interactions among stacked transgenic events that produce changes in efficacy.
Another factor that may add complexity to the trait development process includes enhanced regulatory scrutiny of transgenic crops. Prior to commercialization, the amount of data that can be collected in field studies can be limited by strictly regulated experimental use permits and limited quantities of experimental seed. The regulatory approval process also often requires additional information related to insect resistance monitoring and management and the potential for adverse environmental effects. Collecting such data requires specialized tools and high-precision protocols.
One of the additional regulatory requirements unique to transgenic plant protection mechanisms in the United States is proactive annual monitoring for insect resistance to detect early warning signs indicating resistance development in the field. Detection of resistant insects in these monitoring programs depends, for example, on the level of pest pressure, frequency of resistant individuals, number of samples, and sensitivity of the detection technique. Analytical techniques for resistance monitoring have been developed for Lepidopteran pests and transgenic plants that protect against them. However, new monitoring tools for transgenic plants that protect against other insects, such as soil-inhabiting insects (e.g., corn rootworm) are needed, particularly in view of differing insect life cycles and different sensitivities to insecticidal agents.
Thus, there exists a need for a method of evaluating the efficacy of plant protection mechanisms that minimizes or eliminates the described limitations that may have been encountered with previous studies. Such a method should preferably minimize or eliminate subjective factors and/or the effects of the natural environment and contamination. Such a method should have the predictive power or sensitivity to detect minute or otherwise subtle differences in efficacy while overcoming the limitation of experimental material that is inherent in the transgenic seed product development process. Such a method should be suited to the biology of the target pest and be sufficiently flexible to allow research and regulatory questions to be efficiently addressed while minimizing concerns related to the permits that may be necessary to conduct such efficacy studies.